None of the following discussion of the background of the invention, which is provided solely to aid the reader in understanding the invention, is admitted to be or to describe prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of tyrosine residues on proteins. The phosphorylation state of a protein is modified through the reciprocal actions of tyrosine phosphatases (TPs) and tyrosine kinases (TKs), including receptor tyrosine kinases and non-receptor tyrosine kinases.
RTKs are composed of at least three domains: an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic catalytic domain that can phosphorylate tyrosine residues. The intracellular, cytoplasmic, non-receptor protein tyrosine kinases do not contain a hydrophobic transmembrane domain or an extracellular domain and instead contain non-catalytic domains in addition to their catalytic kinase domains. Such non-catalytic domains include the SH2 domains and SH3 domains. The non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction.
FAK (focal adhesion kinase) and PYK2 (proline-rich tyrosine kinase also known as RAFTK, CAK and CADTK (Lev, et al. (1995) Nature 376:737-745; Avraham, et al. (1995) J. Biol. Chem. 270:27742-27751; Sasaki, et al. (1995) J. Biol. Chem. 270:21206-21219; Yu, et al. (1996) J. Biol. Chem. 271:29993-29998) comprise one family of protein tyrosine kinases. FAK and PYK2 exhibit approximately 45% amino acid sequence identity to each other and each contain an N terminus with similarity to band 4.1 homology domain, a centrally located protein tyrosine kinase domain (Girault, et al. (1999) Trends Neurosci. 22:257-263), and two proline rich regions at the C-terminus (Lev, et al. (1995) Nature 376:737-745). PYK2 and FAK bind to proteins that have been shown to interact with the cytoskeleton such as paxillin (Salgia, et al. (1996) J. Biol. Chem. 271:31222-31226), p130cas, the rhoGAP protein Graf (Ohba, et al. (1998) Biochem. J. 330:1249-1254) and a novel protein containing a LIM domain (Matsuya, et al. (1998) J. Biol. Chem. 273:1003-1014; Lipsky, et al. (1998) J. Biol. Chem. 273:11709-11713).
PYK-2 is a non-receptor tyrosine kinase that is activated by binding of ligand to G-coupled protein receptors such as bradykinin and acetylcholine. PYK2 has a predicted molecular weight of 111 kD and contains five domains: (1) a relatively long N-terminal domain; (2) a kinase catalytic domain; (3) a proline rich domain; (4) another proline rich domain; and (5) a C-terminal domain.
PYK2 is expressed in various tissues, including neural tissues, hematopoietic cells, and in some tumor cell lines. PYK2 is believed to regulate the activity of potassium channels in response to neurotransmitter signaling. PYK2 may also regulate ion-channel function by tyrosine phosphorylation.
PYK2 is activated by stimulation of G-protein coupled receptors (Lev, et al. (1995) Nature 376:737-745), by stimulation of antigen receptors on T cells (Qian, et al. (1997) J. Exp. Med. 185:1253-1259); B cells (Astier, et al. (1997) J. Biol. Chem. 272:228-232), and mast cells (Okazaki, et al. (1997) J. Biol. Chem. 272:32443-32447); as well as in response to inflammatory cytokines (Tokiwa, et al. (1996) Science 273:792-794; Miyazaki, et al. (1998) Genes Dev. 12:770-775; Takaoka, et al. (1999) EMBO J. 18:2480-2488), and stress signals (Tokiwa, et al. (1996) Science 273:792-794). In some cells, tyrosine phosphorylation and activation of PYK2 was shown to be triggered by integrin-mediated adhesion (Astier, et al. (1997) J. Biol. Chem. 272:228-232). Furthermore, PYK2 can be activated by phorbol ester (PMA), or by a variety of extracellular signals that elevate intracellular Ca+2 concentration (Lev, et al. (1995) Nature 376:737-745).
It has been proposed that PYK2 acts in concert with Src to link Gi or Gq coupled receptors with the MAP kinase signaling pathway (Dikic, et al. (1996) Nature 383:547-550). Autophosphorylation of Y402 on PYK2 generates a binding site for the SH2 domain of the docking protein Grb2, and subsequent recruitment of the Grb2/Sos complex leads to activation of the Ras/MAP kinase signal transduction cascade (Dikic, et al. (1996) Nature 383:547-550). Activation of the JNK pathway can also be mediated by PYK2, and this signaling pathway can be inhibited by dominant-negative forms of rac and cdc42 (Tokiwa, et al. (1996) Science 273:792-794). In addition, PYK2 activation leads to suppression of outward potassium currents via tyrosine phosphorylation of the delayed rectifier-type potassium channel Kv1.2 (Lev, et al. (1995) Nature 376:737-745). PYK2 also interacts with and phosphorylates a family of phosphatidylinositol transfer proteins, designated Nirs (Lev, et al. (1999) Mol. Cell. Biol. 19:2278-2288), and with a novel ArfGAP, designated Pap (Andreev, et al. (1999) Mol. Cell. Biol. 19:2338-2350), both in vitro and in vivo. In addition, it was shown that activation of PYK2 leads to tyrosine phosphorylation of other proteins that play a role in signal transmission, including the adaptor proteins Shc (Lev, et al. (1995) Nature 376:737-745) and Cas (Astier, et al. (1997) J. Biol. Chem. 272:228-232).
PYK2 is activated by extracellular signals that lead to calcium influx or calcium release from internal stores. PYK2 is phosphorylated on tyrosine residues in response to a variety of external stimuli. PYK2 may provide a link between G-protein coupled receptors and calcium influx and the MAP kinase signaling pathway, a pathway that relays signals from the cell surface to regulate transcriptional events in the nucleus.
In the PCT Publication WO 98/07870 (Avraham, et al.), the authors discuss PYK2 and state that                “. . . RAFTK therapeutics which modulate RAFTK activity in B cells, T cells, and monocytes can be used to treat immune-mediated disorders and mediate both cell mediated and humoral immune responses.        Normal hematopoietic cells are dependent on growth factors for growth and differentiation and the loss of this growth factor dependence can lead to autonomous growth. The involvement of RAFTK in several growth factor signaling pathways indicates that misse[x]pression of RAFTK can lead to the development of cancers, and the present invention contemplates modulating RAFTK expression and/or activity to control aberrant cell growth. In a preferred embodiment RAFTK is modulated to treat cancers of hematopoietic cells. In another embodiment malignancy can be suppressed in certain cells e.g., leukemic cells, by modulating RAFTK to induce cellular differentiation . . .        . . . In one embodiment the RAFTK proteins of the present invention can modulate the differentiation or maturation of hematopoietic cells; the subject RAFTK polypeptides are capable of influencing both the differentiation and maturation of pluripotent stem cells and the proliferation of differentiated cells.”        